TEM1-beta-Lactamase/beta-lactamase Inhibitor Protein (BLIP)



The enzyme TEM1 β-lactamase (EC 3.5.2.6; TEM1) and its protein inhibitor, β-lactamase inhibitor protein (BLIP) form a complex. Overlap of the residues which participate in TEM1-BLIP interactions between: 1) complex of mutated BLIP (F142A) and wildtype TEM1 (lime; 1s0w) ; 2) complex of the wildtype BLIP/wildtype TEM1 (magenta; 1jtg); 3) unbound wildtype BLIP (structure from Ref 2) and unbound wildtype TEM1 (1btl), these two structures colored orange ). T marks residues of TEM1 and B of BLIP. The distance between TEM Glu-104 and BLIP Lys-74 is marked for the wildtype (1jtg) and mutated (F142A, 1s0w) TEM1-BLIP complex structures (Refs 1, 2).

The TEM1–BLIP in the KFYEY mutant structure (1xxm) is overlaid on the wildtype complex (1jtg) structure. Mutated TEM1 is shown in red, BLIP (lime) (1xxm), wildtype TEM1 (yellow) and BLIP (1jtg) in  orange , respectively. Mutated BLIP residues (K74A, F142A, Y143A) are colored in blue-violet, TEM1 residues E104 and <font color='blue'>Y105 , where mutations to alanines were performed (i.g. E104A, Y105A), are colored <font color='blue'>blue and corresponding to them alanines <font color='magenta'>A104 and <font color='magenta'>A105 are colored <font color='magenta'>magenta in the multiple mutant complex (KFYEY). These two structures are very similar. All-atom RMS deviation (RMSD) between the structures of the wildtype and the KFYEY mutant is 0.37 Å (Ref 1).

The <scene name='2b5r/Bound_unbound/3'>TEM1-BLIP complex where BLIP residues of the <font color='blue'>bound structure are colored <font color='blue'>blue and of the <font color='cyan'>unbound are in <font color='cyan'>cyan. TEM1 residues from the <font color='red'>bound complex are in <font color='red'>red and from the <font color='black'>unbound structure in yellow. <font color='blue'>BLIP and <font color='red'>TEM1 residues are labeled <font color='blue'>blue and <font color='red'>red. The <scene name='2b5r/Bound_unbound/4'>interface between BLIP and TEM1 was divided into six interface clusters (<scene name='2b5r/C1/2'>C1, <scene name='2b5r/C2/3'>C2 , <scene name='2b5r/C3/2'>C3 , <scene name='2b5r/C4/3'>C4 , <scene name='2b5r/C5/2'>C5 , <scene name='2b5r/C6/2'>C6 ). Superpositions of these clusters from TEM1–BLIP (complex-1jtg), TEM1 (unbound-1btl) and BLIP (unbound, from Ref 2) structures are shown. <scene name='2b5r/C1mut/2'>Overlap of cluster C1 from TEM1-BLIP wt, mutant and unbound structures (<font color='red'>TEM1wt -<font color='blue'>BLIPwt complex, <font color='lime'>TEM1wt-BLIPD49A, <font color='black'>unbound TEM1 (yellow), and <font color='cyan'>unbound BLIP ). <font color='blue'>D49 in BLIP is located in the center of C1, surrounded by <font color='red'>4 TEM1 residues. The <font color='blue'>D49A mutation (i.e. removal of a side chain) does not cause structural change in the TEM1-BLIP complex.

To analyze the contribution of non-alanine mutations, E104Y and Y105N in the TEM1 protein were constructed. These residues are polar and have a similar size, hence no major structural changes are expected for these mutants. The complex structure of TEM1 E104Y-Y105N (TEM1YN) with BLIPwt was solved (2b5r). The YN mutation caused only small reduction of binding energy (4.2 kJ/mol), which is slightly less then that obtained for the alanine substitutions of these residues. The <scene name='2b5r/Superpos/1'>superposition of <font color='magenta'>TEM1wt -<font color='black'>BLIPwt (yellow) complex (1jtg; <font color='magenta'>TEM1 colored in magenta; <font color='black'>BLIP in yellow ) with <font color='lime'>TEM1YN (colored in lime) - <font color='pink'>BLIPwt (pink) complex (2b5r). Only a <scene name='2b5r/Superposition/12'>minor change <font color='orange'>(colored orange) in <scene name='2b5r/Superpos/2'>TEM1YN was observed. But, these small changes in TEM1 sequence lead to a major local <scene name='2b5r/Blip/3'>backbone rearrangement of the <scene name='2b5r/Blip/4'>hairpin loop between <font color='blue'>residues 46–53 in <font color='black'>BLIP (yellow). This rearrangement of the <font color='red'>loop is not observed in the <font color='pink'>unbound BLIP structure. This shows that the rearrangement of BLIP was caused by the TEM1 mutations, resulting in a new low energy state. <scene name='2b5r/Superpos/3'>Superposition of residues in wt and mutant complexes near the BLIP 46-53 loop. TEM1 <font color='cyan'>wt E104 and Y105 are colored cyan, while <font color='orange'>mutant E104Y and Y105N are colored orange. BLIP residues colored in <font color='blue'>blue and <font color='red'>red in the <font color='blue'>wt and <font color='red'>mutant complexes (Ref 3).

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